ABSTRACT The ubiquitin-proteasome system has been shown to play an important role in the replication cycle of different viruses. In this study, we describe a strong impairment of rotavirus replication upon inhibition of proteasomal activity. The effect was evidenced at the level of accumulation of viral proteins, viral RNA, and yield of infective particles. Kinetic studies revealed that the early steps of the replicative cycle following attachment, entry, and uncoating were clearly more sensitive to proteasome inhibition. We ruled out a direct inhibition of the viral polymerase activities and stability of viral proteins and found that the crucial step that was impaired by blocking proteasome activity was the assembly of new viroplasms. This was demonstrated by using chemical inhibitors of proteasome and by gene silencing using small interfering RNAs (siRNAs) specific for different proteasomal subunits and for the ubiquitin precursor RPS27A. In addition, we show that the effect of proteasome inhibition on virus infection is not due to increased levels of beta interferon (IFN-β).